A wide variety of proteins in neuronal, endocrine and immune tissues undergo proteolytic processing. Many of these proteins and peptides are intercellular messengers. Most neuroendocrine peptides are synthesized from precursor proteins. Post-translational processing of these precursors is a key step in the production of biologically active peptides. In the majority of cases this occurs by proteolysis of the precursors at 'classical' cleavage sites involving endopeptidases of the subtilisin family. A subset of bioactive peptides are generated by processing at 'non-classical' cleavage sites; the protease(s) responsible for these cleavages have not been well explored. Members of the metalloendoprotease family are thought to be involved in non-classical processing. Among them, endothelin converting enzyme-2 (ECE-2) is a good candidate since it exhibits functional properties that are consistent with it being a neuropeptide biosynthetic enzyme. In this grant application we propose to examine the hypothesis that "ECE-2 is involved in the generation of novel neuropeptides by processing at non-classical sites." The specific aims are (1) to examine the cleavage site selectivity of ECE-2 in order to define a consensus motif for recognition by the enzyme, (ii) to determine the cellular and sub cellular localization of ECE-2 and co-localization with neuropeptides, and (iii) to evaluate the potential role of ECE-2 in neuropeptide processing using animals lacking ECE-2. The studies described in these specific aims are highly interrelated and will expand our current knowledge of neuropeptide processing by providing information about additional neuropeptides and their processing enzymes. Furthermore, findings from these studies will form a basis for future research exploring the functions of these novel neuroendocrine peptides in vivo. Since a variety of neuropeptides are involved in the normal functioning of the brain, understanding the mechanisms that regulate their endogenous levels becomes a prerequisite to the development of strategies for the treatment of several neuroendocrine pathologies as well as neurodegenerative disorders. [unreadable] [unreadable]